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Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory.Key points• Pseudomonas putida advances to a global industrial cell factory.• Novel tools enable system-wide understanding and streamlined genomic engineering.• Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs.

Background Cis , cis -muconic acid (MA) is a dicarboxylic acid of recognized industrial value. It provides direct access to adipic acid and terephthalic acid, prominent monomers of commercial plastics. Results In the present work, we engineered the soil bacterium Corynebacterium glutamicum into a stable genome-based cell factory for high-level production of bio-based MA from aromatics and lignin hydrolysates. The elimination of muconate cycloisomerase ( catB ) in the catechol branch of the β-ketoadipate pathway provided a mutant, which accumulated MA at 100% molar yield from catechol, phenol, and benzoic acid, using glucose as additional growth substrate. The production of MA was optimized by constitutive overexpression of catA , which increased the activity of the encoded catechol 1,2-dioxygenase, forming MA from catechol, tenfold. Intracellular levels of catechol were more than 30-fold lower than extracellular levels, minimizing toxicity, but still saturating the high affinity CatA enzyme. In a fed-batch process, the created strain C. glutamicum MA-2 accumulated 85 g L −1 MA from catechol in 60 h and achieved a maximum volumetric productivity of 2.4 g L −1  h −1 . The strain was furthermore used to demonstrate the production of MA from lignin in a cascade process. Following hydrothermal depolymerization of softwood lignin into small aromatics, the MA-2 strain accumulated 1.8 g L −1 MA from the obtained hydrolysate. Conclusions Our findings open the door to valorize lignin, the second most abundant polymer on earth, by metabolically engineered C. glutamicum for industrial production of MA and potentially other chemicals.

Long-chain polyunsaturated fatty acids (LC-PUFAs), particularly the omega-3 LC-PUFAs eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), have been associated with beneficial health effects. Consequently, sustainable sources have to be developed to meet the increasing demand for these PUFAs. Here, we demonstrate the design and construction of artificial PUFA biosynthetic gene clusters (BGCs) encoding polyketide synthase-like PUFA synthases from myxobacteria adapted for the oleaginous yeast Yarrowia lipolytica . Genomic integration and heterologous expression of unmodified or hybrid PUFA BGCs yielded different yeast strains with specific LC-PUFA production profiles at promising yield and thus valuable for the biotechnological production of distinct PUFAs. Nutrient screening revealed a strong enhancement of PUFA production, when cells were phosphate limited. This represents, to the best of our knowledge, highest concentration of DHA (16.8 %) in total fatty acids among all published PUFA-producing Y. lipolytica strains. Sustainable sources are needed to meet the demand for long-chain polyunsaturated fatty acids. Here the authors construct an artificial biosynthetic gene cluster in Y. lipolytica capable of producing a high concentration of PUFAs.

Background Lysine-derived C5 compounds are important intermediates in cellular metabolism and promising building blocks for sustainable polymer chemistry. Among these, 5-aminovalerate (5-AVA) has been extensively studied as a platform chemical produced via a two-step microbial pathway. However, its direct precursor, 5-aminovaleramide (5-AVD), generated from lysine by lysine 2-monooxygenase, remains largely unexplored. Notably, 5-AVD is an attractive product in its own right, as it provides a versatile intermediate for the synthesis of polyamides and other nitrogen-containing chemicals. Here, we establish the first de novo microbial production of 5-AVD by systematically engineering Corynebacterium glutamicum for optimized precursor flux, product export, and redox balance. Results Trace secretion of 5-AVD was discovered in 5-AVA-producing strains, and tolerance studies showed that C. glutamicum can withstand high 5-AVD concentrations. To exploit this trait, the lysine-producing strain LYS-12 was engineered to express the davB gene from Pseudomonas putida under the constitutive tuf promoter, resulting in increased 5-AVD secretion. Pathway analysis revealed that the native exporter LysE is essential for efficient 5-AVD export, while heterologous GABA permeases provided no benefit. Mechanistic analysis further showed that LysE preferentially exports lysine over 5-AVD, establishing it as a flux gatekeeper that critically shapes product selectivity. Overexpression of heterologous NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) enhanced NADPH supply and improved redox balance, increasing the 5-AVD yield to 0.32 mol mol −1 in strain AVD-11. In fed-batch fermentation, AVD-11 reached a maximum productivity of 1.2 g L −1 h −1 and a final titer exceeding 36 g L −1 with > 97% selectivity, while chromosomally integrated davB remained genetically stable throughout the process. Conclusions This study establishes C. glutamicum as a robust and industrially relevant platform for the sustainable production of 5-AVD. By combining rational pathway design, transporter control, and cofactor engineering, we deliver the first high-yield microbial route to this valuable amide and provide a blueprint for expanding the portfolio of lysine-derived monomers accessible through microbial cell factories.

Background Yarrowia lipolytica is an emerging host for producing acetyl-CoA– and malonyl-CoA–derived chemicals. However, most processes rely on yeast nitrogen base (YNB), a historical formulation with poorly controlled trace metal content. This variability impairs metabolic performance, limits reproducibility, and complicates process transfer. Results Commercial YNB batches differed markedly, causing 1.5–2-fold variation in growth and docosahexaenoic acid (DHA) production. We developed a malonyl-CoA–responsive flaviolin reporter strain and combined it with a structured Design of Experiments (DoE) workflow to systematically re-engineer YNB mineral composition. Dissection of all 20 YNB components revealed that vitamins are dispensable under the tested conditions, whereas a small subset of salts and trace elements - particularly ZnSO 4 , FeCl 3 , KH 2 PO 4 , MgSO 4 , CaCl 2 , and CuSO 4 - dominantly shape precursor availability and product formation. One-factor-at-a-time (OFAT), factorial, steepest ascent, and central composite designs converged in an optimized synthetic mineral medium assembled entirely from individual salts and trace metals. This formulation increased flaviolin titers to 1.41 ± 0.08 g L -1 , a more-than threefold improvement over commercial YNB, while ensuring high reproducibility. Key mineral interventions also translated to complex pathways: omission of ZnSO 4 increased PUFA titers by 7.6-fold (docosapentaenoic acid, DPA) and 58-fold (eicosapentaenoic acid, EPA) and enhanced DHA formation in independent production strains. The defined formulation substantially reduces cost and eliminates batch-to-batch variability inherent to commercial YNB powders. Conclusions Our results establish mineral balancing as a major yet underused lever for improving acetyl-CoA– and malonyl-CoA–derived production in Y. lipolytica and demonstrate a generalizable, model-guided workflow for creating simplified, reproducible, and cost-efficient synthetic media for non-conventional yeast cell factories.

Background Long-chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA), are essential for human health and have been widely used in the food and pharmaceutical industries. However, the limited availability of natural sources, such as oily fish, has led to the pursuit of microbial production as a promising alternative. Yarrowia lipolytica can produce various PUFAs via genetic modification. A recent study upgraded Y. lipolytica for DHA production by expressing a four-gene cluster encoding a myxobacterial PKS-like PUFA synthase, reducing the demand for redox power. However, the genetic architecture of gene expression in Y. lipolytica is complex and involves various control elements, offering space for additional improvement of DHA production. This study was designed to optimize the expression of the PUFA cluster using a modular cloning approach. Results Expression of the monocistronic cluster with each gene under the control of the constitutive TEF promoter led to low-level DHA production. By using the minLEU2 promoter instead and incorporating additional upstream activating UAS1B4 sequences, 5' promoter introns, and intergenic spacers, DHA production was increased by 16-fold. The producers remained stable over 185 h of cultivation. Beneficially, the different genetic control elements acted synergistically: UAS1B elements generally increased expression, while the intron caused gene-specific effects. Mutants with UAS1B16 sequences within 2–8 kb distance, however, were found to be genetically unstable, which limited production performance over time, suggesting the avoidance of long repetitive sequence blocks in synthetic multigene clusters and careful monitoring of genetic stability in producing strains. Conclusions Overall, the results demonstrate the effectiveness of synthetic heterologous gene clusters to drive DHA production in Y. lipolytica . The combinatorial exploration of different genetic control elements allowed the optimization of DHA production. These findings have important implications for developing Y. lipolytica strains for the industrial-scale production of valuable polyunsaturated fatty acids.

Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed (“fluxed”) through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents. Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized “EDEMP cycle” (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa ; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed (“fluxed”) through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

Background Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. Results Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl 2 boosted production. The IPTG-inducible producer C . glutamicum CR099 pXMJ19   P tac pedACD Cg provided 66 mg L −1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain P tuf pedACD Cg allowed successful production without the need for IPTG. Conclusions The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.

Background Antimicrobial peptides (AMPs) such as pediocin PA-1 are attractive for food biopreservation and infection control, but their broader use is limited by low recombinant yields and high production costs. Corynebacterium glutamicum has emerged as a robust GRAS chassis for heterologous peptide and protein production, yet commonly used shuttle vectors provide only moderate plasmid copy numbers and expression capacities. In particular, existing pediocin PA-1 processes in C. glutamicum rely on standard pBL1 - or pCG1-family vectors that do not yet leverage replication-origin engineering. Results We rationally redesigned the replication control region of the widely used pClik 5α (pCG1-family) backbone by introducing targeted mutations in the repA gene, an antisense RNA ( cgrI ) promoter, and putative partitioning genes parAB , and constructed a systematic panel of high-copy variants. Using a P tuf -driven mCherry reporter as a quantitative readout, we identified plasmids that supported several-fold higher fluorescence than the parental backbone while maintaining robust growth. Fluorescence-based gene-dosage estimation indicated a strong increase in apparent plasmid copy number. Independent qPCR-based plasmid copy number determination using two plasmid loci confirmed that the lead variant pClik 5α repA mut reached approximately 28–30 copies per chromosome equivalent, compared to approximately 2–3 copies for the parental plasmid, corresponding to an approximately 10-fold increase. Genome-wide transcriptome analysis revealed a defined and adaptive transcriptional response to elevated plasmid copy number and expression burden, characterized by adjustments in membrane-associated transport, respiratory functions, and amino acid-related metabolism, without evidence of collapse of core biosynthetic functions. When the best-performing replicon was applied to episomal expression of a codon-optimized pedACD Cgl operon, pediocin PA-1 titers increased by 2.5-fold compared to the best pXMJ19-based reference under identical, previously optimized process conditions, placing the system, under comparable cultivation formats, within the upper range of reported microbial pediocin production processes. Conclusions This work demonstrates that rational engineering of pCG1-family replication modules in C. glutamicum can unlock markedly higher plasmid copy numbers and expression capacities while preserving physiological robustness. The resulting high-copy pClik 5α derivatives, exemplified by pClik 5α repA mut , provide a versatile high-copy expression platform with demonstrated utility for recombinant reporter protein and antimicrobial peptide production in C. glutamicum and offer a foundation for further integration with folding, secretion, and process engineering strategies to advance industrial AMP production.

Background Extremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing. Results Here , we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia , reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C.   glutamicum Ptuf ectD PST achieved a titre of 74 g L −1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABC opt , was successfully achieved without intermediate purification. Conclusions C. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.